The role of acetylation in Timp‐1 regulation

Abstract

Introduction Aberrant matrix turnover, mediated by a family of proteases known as matrix metalloproteinases (MMPs), is involved in a number of pathologies including rheumatoid arthritis and osteoarthritis, tumour invasion and metastasis, and liver fibrosis. The active forms of all of the MMPs are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). As inhibition represents a major level of control of MMP activity, a detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important in developing therapies for these diseases. Histone acetyl transferases (HATs) play a crucial role in gene regulation via acetylation of both histones (to loosen nucleosomal structure and to recruit accessory factors) and transcription factors. Here, we describe the effects of a histone deacetylase inhibitor (Trichostatin A – TSA) on the TGF‐β1 and Phorbol ester (PMA) induction of Timp‐1.Materials and methods RNA isolation, TaqMan® real‐time RT‐PCR, cell culture, transient transfections and reporter gene assays along with electrophoretic mobility shift assays (EMSA) were performed essentially as described (Young et al. 2002).Results In murine C3H10T1/2 cells both TGF‐β1 and PMA induce Timp‐1 expression in a protein synthesis‐dependent manner as measured by Northern blotting and TaqMan® real‐time RT‐PCR. TSA superinduces PMA‐induced Timp‐1 expression but represses TGF‐β‐induced Timp‐1 expression. A TSA dose–response experiment further demonstrates that TGF‐β and PMA stimulate Timp‐1 gene expression via different mechanisms. The effects of TGF‐β, PMA and TSA on Timp‐1 expression can be reiterated in transient transfection studies with Timp‐1 promoter containing reporter constructs. Deletion of the proximal AP‐1 motif from the promoter demonstrates that this sequence is critical for TGF‐β1‐induced reporter expression, while PMA appears to act via other sequences/mechanisms.Discussion Repression of deacetylation by TSA differentially affects the TGF‐β or PMA induction of Timp‐1. These results will help elucidate the different molecular mechanisms by which these factors regulate the expression of Timp‐1 and other genes. Furthermore, the effects of acetylation on TGF‐β induction of Timp‐1 may act as a paradigm for other cytokine‐induced genes.This work was funded and supported by the Arthritis Research Campaign (ARC) and the Dunhill Medical Trust.